[CV1000] GetClone™ PCR Cloning Vector, 20 Rxn

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Description 

The GetClone™ PCR Cloning Vector is a positive selection system for the high efficiency cloning of blunt end DNA or amplicons. This cloning vector contains a lethal gene which can be disrupted by ligation with a blunt end DNA insert at the cloning site. After ligation and transformation, only E.coli clones carrying the pGet1.1 Vector with inserted DNA at cloning site are able to propagate on LB-ampicillin agar plates, eliminating the additional needs of IPTG and X-Gal for blue/ white screening. 


Features

  • Cloning efficiency was greater than 90% 

  • The IPTG and X-Gal are not required 

  • Accepts a wide range of insert/ vector ratio 0.5:1 to 12:1 

  • Accepts insert size from 6 bp to 12 kb

  • The phosphorylation of PCR fragments is not required

  • Accepts blunt end amplicon or DNA fragment (not for sticky end)


Storage

-20°C for 24 months

Odoo - Sample 1 for three columns

The plasmid map of pGet1.1 Vector 


Odoo - Sample 2 for three columns

The plasmid cloning sites of pGet1.1 Vector

Contents

Component

Volume

pGet1.1 Vector (50 ng/μl)

23 μl

pGet-For Primer (10 μM)

100 μl

pGet-Rev Primer (10 μM)

100 μl


Primers Sequence                       

pGet-For Primer:               

5'-TCGAAGTTAAAGATGATTACGG-3'

pGet-Rev Primer:

5'-TCTCTCGATAGCATTTCCTGC-3'


Storage

 -20°C for 24 months

 



Ligation Example 1 (NEB T4 DNA Ligase #M0202)


Insert (Blunt end)       X μl (Y ng*)

pGet1.1(2995 bp)       1 μl (50 ng)

Mix well then add

 

10X T4 DNA Ligase Buffer  2 μl

T4 DNA Ligase                    1 μl

ddH2O                           to 20 μl

Final volume                    20 μl

Mix well then incubate at 16°C or room temperature (20~25°C) for 1 hours.

 

Ligation Example 2 (TOYOBO Ligation High ver2 #LGK-201)


Insert (Blunt end)       X μl (Y ng*)

pGet 1.1 (2995 bp)    1 μl (50 ng)

ddH2O                          up to 7 μl

Ligation high ver2               3.5 μl

Final volume                     10.5 μl

Mix well then incubate at 16°C or room temperature (20~25°C) for 5~30 mins.

 

*For 3/1 of Insert/Vector molar ratio:

  


Transformation

The GetClone™ is compatible with most available competent E. coli cells. Apply 1 ~10 µl of ligation mixture to 10 times volume competent E. coli cells. Perform transformation procedures according to the instruction of the competent cells. Spread the transformed E. coli cells on an LB-ampicillin (50~100 µg/ml) plate for colony selection.


Recommended colony PCR condition

(SMOBIO’s TP1200 ExcelTaq™ 5X PCR Master Dye Mix is suggested)

Template

Single colony

pGet-For Primer

0.5 µl

pGet-Rev Primer

0.5 µl

5X PCR Master Mix

5 µl

H2O

to 25 µl

Total volume

25 µl

 

Recommended PCR Program

Steps

Temp.

Time

Cycles

Template denature

94°C

2 min

1

Denature

94°C

30 sec

25-40

Annealing

50°C

30 sec

Extension

72°C

30 sec/kb

Final extension

72°C

1 min

1

 

Odoo - Sample 1 for three columns

High Fidelity PCR amplification

Amplification of target gene with HiFi™ DNA polymerase to minimize error rate.

Odoo - Sample 2 for three columns

Gel electrophoresis

Staining amplicons with safe fluorescent dyes, following by observation under blue-light illuminator to minimize damage of DNA amplicons and maximize successful cloning efficiency.   

Safe fluorescent dyes

Blue-light illuminator

Odoo - Sample 3 for three columns

Transformation

Prepare competent cells with high efficiency and transform with time-saving protocol.  

Odoo - Sample 3 for three columns

Colony PCR

Analyze colonies with PCR master mix to save preparation time.